In mammals, the nucleolus is thought to be a stress sensor; upon cellular stress conditions, a release of nucleolar proteins and down-regulation of rDNA transcription occurs. Since yeast Rrn3p is a homolog of the mammalian RNA polymerase I (Pol I)-specific transcription factor TIF-IA, we decided to investigate the role of Rrn3p in oxidant-induced nucleolar stress in yeast. We show that, after oxidant treatment, the level of Rrn3p is unaffected but Rrn3p is translocated from the nucleolus into the cytoplasm and a point mutation in the RRN3 gene leads to hypersensitivity of the yeast to oxidants. This hypersensitivity can be abolished by re-introduction of the active RRN3 gene, antioxidant supplementation and anoxic atmosphere. Additionally, we employed the PRINS technique to monitor oxidant-mediated changes in the nucleolar structure. Taken together, our results suggest the role of the yeast nucleolus in the response to oxidative stress signals.